A non-starch polysaccharide (NSCYP) isolated and purified from Chinese yam (Dioscorea opposita Thunb.) with its immunomodulatory activities on RAW 264.7 macrophages in vitro was reported. Chemical studies showed that the average molecular weight was 71.78 kDa. NSCYP was mainly composed of mannose, galacturonic acid, glucose, galactose, and arabinose with a molar ratio of 1.09:0.51:1.0:3.03:1.77. Moreover, t-Araf, 1,5-Araf, t-Glcp, t-Galp, t-GalAp, t-GalMEp, 1,4-Galp, 1,4-GalAp, 1,4-GalMEp, 1,4-Glcp, 1,6-Galp, 4,6-Galp and 4,6-Manp were detected in NSCYP. NSCYP promoted RAW 264.7 macrophages phagocytic activity, NO release, transcription and production of interleukin (IL)-6 and tumor necrosis factor (TNF)-α. NSCYP induced NF-κB activation and IκBα degradation in RAW 264.7 cells. Notably, inhibition of Toll-like receptor 4 led to the suppression of NSCYP-induced NO production in RAW 264.7 cells. These findings suggested that NSCYP exhibit the potential as functional food or therapeutic stimulant as a natural immune-stimulating polysaccharide.